DESCRIPTION (Investigator's Abstract): The broad and long-term objective of the proposed research is to use mass spectrometry to study the dynamics of the interactions between metal ions and proteins under conditions which closely resemble the in vivo environment. The diversity of the chemical functionality of proteins provides a multiplicity of sites for metal ion binding. Some of these interactions are part of the natural function of the protein, imparting catalytic activity, conformational change, stability, regulatory function while other interactions are deleterious to its function often resulting in a pathogenic response. These interactions are complex and are influenced by the variables of solution and interfacial thermodynamics and kinetics. Few probes exist that probe these interactions with minimal perturbation. Electron paramagnetic resonance (EPR) comes the closest and much of what the investigators know about the interaction of a metal ion with a protein at the molecular level comes from the use of this technique [1]. The natural interaction of metal ions with a peptide or protein may involve a coordination of a single metal ion as in the case of the interaction of Zn+2/angiotensin or a multiplicity of metal ions or protein molecules as found in Zn+2-coupled oligomers if insulin, Zn+2/metallothioneins or the mixed metal/sulfur clusters imbedded in the structure of nitrogenase. Metal binding sites, the strength of the interaction, conformational changes, the thermodynamics and kinetics of the coordination, the dependence of the interaction on the nature of the metal ion, and influence on biological activity are some of the subjects that are under investigation in this field [2]. Mass spectrometry has played a very small role but recent developments may give this technique more prominence. The aim of this proposal is to develop the technology of 252Cf-plasma desorption mass spectrometry (252Cf-PDMS) and matrix-assisted laser desorption mass spectrometry (MALD) with a focus on applications to the study of the dynamics of metal ion/protein interactions. The following specific aims have been established: to improve the performance of 252Cf-PDMS and MALD for the molecular weight determination of metalloproteins in the mass range of 10,000 - 250,000 D and to develop protocols for studying metal ion/peptide interactions in solution and at solution/solid surface interfaces by 252Cf-PDMS. A fundamental hypothesis to be tested is whether the fragmentation patterns observed in 252Cf-PDMS can be used to identify metal ion binding sites, detect conformational changes and assess the relative strength of the binding. After these protocols have been established, a set of histidine and cystine containing peptides will be selected as model compounds for the study of their interactions with a selection of metal ions that represent natural and unnatural substrates.